Since restenosis after angioplasty still remains a major clinical problems, inhibition of neointimal formation is an important subject. In this study, we focused on the transcription factor, E2F, that plays a pivotal role in the transactivation of cell-cycle regulatory genes, and also we developed a newly delivery system of decoy oligodeoxynucleotides (ODN). We transfected E2F decoy ODN mixed with an echo-contrast microbubble agent (Optison) into rat carotid artery balloon-injured model by using therapeutic ultrasound (US) to inhibit neointimal formation. Two weeks after transfection, the intimal to medial area ratio in E2F decoy + Optison + US group was significantly decreased (P < 0.01). Inhibition of cell growth was also confirmed by PCNA staining. No apparent toxicity such as inflammation could be detected in blood vessels transfected with E2F decoy ODN with Optison and ultrasound. Overall, the present studies demonstrated a novel non-viral ODN transfer method into blood vessels. A novel therapeutic strategy using E2F decoy ODN with Optison using ultrasound may be useful to inhibit restenosis in clinical practice without a viral vector. (C) 2004 Elsevier Inc. All rights reserved.